Alpha-Glucan, Water Dikinase and the Control of Starch Degradation in Arabidopsis thaliana Leaves
This thesis examines the role of alpha-glucan, water dikinase (GWD) in the control of starch degradation in Arabidopsis thaliana leaves. Starch is degraded at night in Arabidopsis leaves to provide carbon for metabolism and growth. The rate of starch degradation is constant through the night and is precisely regulated such that starch reserves last until dawn. Despite the importance of this regulation for normal plant growth, we do not know which enzymes of the pathway are regulated to modulate flux. In this work I focus on the first enzyme in the starch degradation pathway, GWD. This enzyme phosphorylates the starch granule surface, thereby stimulating starch degradation by hydrolytic enzymes. It is hypothesised that GWD is important for the control of starch degradation. GWD shows large diel oscillations in transcript abundance. The enzyme is redox responsive in vitro, and is phosphorylated and SUMOylated in vivo. My work revealed that GWD protein levels are essentially invariant over the diel cycle. Studying plants in which GWD transcript was targeted by inducible RNAi and rapidly decreased to low levels revealed that GWD has half life of 2 days and a very low flux control coefficient for starch degradation. Taken together these findings demonstrate that large diel oscillations in GWD transcript abundance are not important for the daily control of starch degradation. Using transgenic plants in which native GWD was replaced with mutant forms of the enzyme, I demonstrate that redox responsive properties and certain SUMOylation and phosphorylation sites of GWD are not of in vivo relevance for appropriately timed starch degradation. Finally, I identify a previously unknown role for GWD in starch synthesis and find ROC4 to be a novel, putative GWD interaction partner.
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